T cell-redirecting antibody for treatment of solid tumors via targeting mesothelin.

T cell engaging bispecific antibodies (TCBs) have recently become significant in cancer treatment. In this study we developed MSLN490, a novel TCB designed to target mesothelin (MSLN), a glycosylphosphatidylinositol (GPI)-linked glycoprotein highly expressed in various cancers, and evaluated its efficacy against solid tumors. CDR walking and phage display techniques were used to improve affinity of the parental antibody M912, resulting in a pool of antibodies with different affinities to MSLN. From this pool, various bispecific antibodies (BsAbs) were assembled. Notably, MSLN490 with its IgG-[L]-scFv structure displayed remarkable anti-tumor activity against MSLN-expressing tumors (EC50: 0.16 pM in HT-29-hMSLN cells). Furthermore, MSLN490 remained effective even in the presence of non-membrane-anchored MSLN (soluble MSLN). Moreover, the anti-tumor activity of MSLN490 was enhanced when combined with either Atezolizumab or TAA × CD28 BsAbs. Notably, a synergistic effect was observed between MSLN490 and paclitaxel, as paclitaxel disrupted the immunosuppressive microenvironment within solid tumors, enhancing immune cells infiltration and improved anti-tumor efficacy. Overall, MSLN490 exhibits robust anti-tumor activity, resilience to soluble MSLN interference, and enhanced anti-tumor effects when combined with other therapies, offering a promising future for the treatment of a variety of solid tumors. This study provides a strong foundation for further exploration of MSLN490's clinical potential.

A novel bispecific antibody drug conjugate targeting HER2 and HER3 with potent therapeutic efficacy against breast cancer.

Antibody drug conjugate (ADC) therapy has become one of the most promising approaches in cancer immunotherapy. Bispecific targeting could enhance the efficacy and safety of ADC by improving its specificity, affinity and internalization. In this study we constructed a HER2/HER3-targeting bispecific ADC (BsADC) and characterized its physiochemical properties, target specificity and internalization in vitro, and assessed its anti-tumor activities in breast cancer cell lines and in animal models. The HER2/HER3-targeting BsADC had a drug to antibody ratio (DAR) of 2.89, displayed a high selectivity against the target JIMT-1 breast cancer cells in vitro, as well as a slightly higher level of internalization than HER2- or HER3-monospecific ADCs. More importantly, the bispecific ADC potently inhibited the viability of MCF7, JIMT-1, BT474, BxPC-3 and SKOV-3 cancer cells in vitro. In JIMT-1 breast cancer xenograft mice, a single injection of bispecific ADC (3 mg/kg, i.v.) significantly inhibited the tumor growth with an efficacy comparable to that caused by combined injection of HER2 and HER3-monospecific ADCs (3 mg/kg for each). Our study demonstrates that the bispecific ADC concept can be applied to development of more potent new cancer therapeutics than the monospecific ADCs.

Thyroid hormone attenuates vascular calcification induced by vitamin D3 plus nicotine in rats.

Thyroid hormones (THs) including thyroxine (T4) and triiodothyronine (T3) play critical roles in bone remodeling. However, the role and mechanism of THs in vascular calcification (VC) have been unclear. To explore the pathophysiological roles of T3 on VC, we investigated the changes in plasma and aortas of THs concentrations and the effect of T3 on rat VC induced by vitamin D3 plus nicotine (VDN). VDN-treated rat showed decreased plasma T3 content, increased vascular calcium deposition, and alkaline phosphatase (ALP) activity. Administration of T3 (0.2 mg/kg body weight IP) for 10 days greatly reduced vascular calcium deposition and ALP activity in calcified rat aortas when compared with controls. Concurrently, the loss of smooth muscle lineage markers α-actin and SM22a was restored, and the increased bone-associated molecules, such as runt-related transcription factor2 (Runx2), Osterix, and osteopontin (OPN) levels in calcified aorta, were reduced by administration of T3. The suppression of klotho in calcified rat aorta was restored by T3. Methimazole (400 mg/L) blocked the beneficial effect of T3 on VC. These results suggested that T3 can inhibit VC development.

Cinchona alkaloids from Cinchona succirubra and Cinchona ledgeriana.

Seven new cinchona alkaloids, cinchonanines A-G (1-7), and 29 known alkaloids were isolated from the barks of Cinchona surrirubra and C. ledgeriana collected from Yunnan Province in China. The new structures were elucidated by extensive spectroscopic analysis. All compounds were evaluated for their cytotoxicity against five human cancer cell lines. Compounds 2, 13, 14, and 15 showed moderate cytotoxicity.

Melosuavines A-H, cytotoxic bisindole alkaloid derivatives from Melodinus suaveolens.

Eight new bisindole alkaloids, melosuavines A-C (1-3), having an aspidosperma-scandine linkage, melosuavines D-F (4-6), possessing an aspidosperma-aspidosperma skeleton, and melosuavines G and H (7 and 8) of the aspidosperma-venalatonine type, tenuicausine (9), and melodinine J (10) were isolated from the twigs and leaves of Melodinus suaveolens. The structures of 1-8 were elucidated by extensive spectroscopic methods, and compounds 9 and 10 were identified by comparison with data in the literature. The relative configuration 9 was determined from the ROESY spectrum, and some NMR signals were reassigned. Compounds 1, 2, 4-6, 8, and 10 exhibited low micromolar cytotoxicity against one or more of five human cancer cell lines.

Intermedin1-53 attenuates vascular smooth muscle cell calcification by inhibiting endoplasmic reticulum stress via cyclic adenosine monophosphate/protein kinase A pathway.

We previously reported that endoplasmic reticulum (ER) stress-mediated apoptosis participated in vascular calcification. Importantly, a novel paracrine/autocrine peptide intermedin1-53 (IMD1-53) in the vasculature inhibited vascular calcification in rats. But the mechanisms needed to be fully elucidated. Vascular smooth muscle cells (VSMCs) calcification was induced by CaCl2 and ß-glycerophosphate. Tunicamycin (Tm) or dithiothreitol (DTT) was used to induce ER stress. We found that IMD1-53 (10(-7)mol/L) treatment significantly alleviated the protein expression of ER stress hallmarks activating transcription factor 4 (ATF4), ATF6, glucose-regulated protein 78 (GRP78) and GRP94 induced by Tm or DTT. ER stress occurred in early and late calcification of VSMCs but was inhibited by IMD1-53. These inhibitory effects of IMD1-53 were abolished by treatment with the protein kinase A (PKA) inhibitor H89. Pretreatment with IMD1-53 decreased the number of apoptotic VSMCs and downregulated protein expression of cleaved caspase 12 and C/EBP homologous protein (CHOP) in calcified VSMCs. Concurrently, IMD1-53 restored the loss of VSMC lineage markers and ameliorated calcium deposition and alkaline phosphatase activity in calcified VSMCs as well. The observation was further verified by Alizarin Red S staining, which showed that IMD1-53 reduced positive red nodules among calcified VSMCs. In conclusion, IMD1-53 attenuated VSMC calcification by inhibiting ER stress through cAMP/PKA signalling.

Gardovatine, a novel Strychnos-Strychnos bisindole alkaloid with cytotoxicity from Gardneria oveta.

Gardovatine (1), the first Strychnos-Strychnos alkaloid with a C3/C7 cleaved backbone, was isolated from twigs and leaves of Gardneria ovate, together with an analogue divarine (2). The structure was established by extensive spectroscopic methods. Both compounds showed potential cytotoxicities against five human cancer cell lines.

Transforming growth factor-ß1 gene mutations and phenotypes in pediatric patients with Camurati­Engelmann disease.

The aim of the present study was to investigate the clinical characteristics and major causative gene in pediatric patients with Camurati­Engelmann disease (CED). Biochemical and radiographic examinations, bone scintigraphy and genetic analyses were performed in two affected males and their parents. The two patients experienced waddling gait, muscular weakness and growth developmental delay. X-ray radiography revealed typical fusiform thickening of the diaphyseal portions of the long bones. The abnormal uptake of tracer Tc-99m was visualized in the skull and both sides of the upper humeri, ulnas, radii, femurs and tibias using bone scintigraphy. Serum levels of the bone formation marker procollagen type I N-terminal propeptide (PINP) and the bone resorption marker ß­isomerized C-terminal cross-linked telopeptide of type I collagen (ß-CTX) in the 6-year-old patient were significantly increased compared with the normal value range, while only the ß-CTX levels were elevated in the 16-year-old patient. A heterozygous missense mutation p.Arg218Cys in exon 4 of the transforming growth factor ß1 (TGFß1) gene was detected in the two patients, while their parents had normal wild­type genotypes. In conclusion, the p.Arg218Cys mutation was shown to contribute to the clinical phenotypes in two pediatric patients with CED. The results of this study suggest that abnormal bone turnover marker levels, typical radiological findings and mutations in the TGFß1 gene are three important factors in the diagnosis of sporadic CED cases.

Angiotensin-(1-7) inhibits vascular calcification in rats.

Angiotensin-(1-7) [Ang-(1-7)] is a new bioactive heptapeptide in the renin-angiotensin-aldosterone system (RAAS) with potent protective effects in cardiovascular diseases, opposing many actions of angiotensin II (Ang II) mediated by Ang II type 1 (AT1) receptor. It is produced mainly by the activity of angiotensin-converting enzyme 2 (ACE2) and acts through the Mas receptor. However, the role of Ang-(1-7) in vascular calcification (VC) is still unclear. In this study, we investigated the protective effects of Ang-(1-7) on VC in an in vivo rat VC model induced by vitamin D3 plus nicotine. The levels of ACE2 and the Mas receptor, as well as ACE, AT1 receptor, Ang II type 2 receptor and angiotensinogen, were significantly increased in calcified aortas, and Ang-(1-7) reversed the increased levels. Ang-(1-7) restored the reduced expression of lineage markers, including smooth muscle (SM) α-actin, SM22α, calponin and smoothelin, in vascular smooth muscle cells (VSMCs) and retarded the osteogenic transition of VSMCs by decreasing the expression of bone-associated proteins. It reduced alkaline phosphatase activity and calcium deposition in VC and alleviated the hemodynamic disorders of rats with VC. We provide the first in vivo evidence that Ang-(1-7) can inhibit the development of VC by inhibiting the osteogenic transition of VSMCs, at least in part by decreasing levels of the ACE/Ang II/AT1 axis. The increased expression of ACE2 and the Mas receptor in calcified aortas suggests the involvement of the ACE2/Ang-(1-7)/Mas axis during VC. Ang-(1-7) might be an efficient endogenous vasoprotective factor for VC.

[The relationship between hyperuricaemia and clinic pathology of IgA nephropathy].

OBJECTIVE: To analyze the correlation between the level of serum uric acid and the clinical and pathological features of IgA nephropathy. METHODS: Totally 148 patients diagnosed as IgA nephropathy by renal biopsy in our hospital from January 2007 to December 2010 were divided into hyperuricaemic group (41 cases) and non-hyperuricaemic group (107 cases) according to the level of serum uric acid. The clinical parameters and renal pathology grade were compared. RESULTS: There were significant differences between hyperuricaemic group and non-hyperuricaemic group in the incidences of hypertension (63.4% vs 38.3%), disease duration [(18.90 ± 10.12) months vs (9.46 ± 3.91) months] and body mass index [(22.81 ± 3.60) kg/m(2) vs (15.32 ± 2.54) kg/m(2)] (all P < 0.05), while no differences in age and sex (both P > 0.05). The blood urea nitrogen (BUN) [(8.93 ± 4.28) mmol/L vs (5.21 ± 2.18) mmol/L], creatinine (Cr) [(155.96 ± 107.72) µmol/L vs (79.52 ± 40.01) µmol/L], serum triglycerides [(2.11 ± 1.06) mmol/L vs (1.86 ± 1.20) mmol/L] and 24-hour urine protein amount [(4328.16 ± 1434.25) mg/24 h vs (2885.10 ± 1388.15) mg/24 h] were significantly different between the two groups (all P < 0.05). The percentage of Lee's grade I + II in hyperuricaemic group was 12.2%, and IV + V grade was 39.0%, while percentage of Lee's grade I + II in non-hyperuricaemic group was 25.2%, and IV + V grade was 16.9% (P < 0.05). Tubulointerstitial lesions (TIL) grade III + IV was more in hyperuricaemic group, which was 68.3%, while TIL grade II was more in non-hyperuricaemic group, which was 76.6%. Renal artery damage grade II + III was more in hyperuricaemic group, which was 73.2%, while renal artery damage grade 0 + I was more in non-hyperuricaemic group, which was 69.2%. CONCLUSIONS: The level of serum uric acid was related with 24-hour urine protein amount, blood pressure and kidney function in IgA nephropathy, and Lee's grade, TIL grade and renal artery damage grade were severe in hyperuricaemic group.

Effect and mechanism of nociceptin/orphanin FQ reversing multi-drug resistance in K562/ADM cell.

OBJECTIVE: To investigate the effect and mechanism of nociceptin/orphanin FQ (OFQ) reversing multi-drug resistance of K562/ADM cells in vitro. METHODS: MTT assay, Wright staining, flow cytometry, transmission electron microscope and gel electrophoresis were used to evaluate the effect and mechanism of OFQ in reversing multi-drug resistance of K562/ADM cells. RESULTS: OFQ could time-dependently reverse the ADM resistance of K562/ADM cell. After treatment with OFQ (1 x 10(-7) mol x L(-1)), K562/ADM cells were cultured for 24, 48 and 72 h. The reversal index (RI) was 1.33, 1.42 and 1.53, respectively. Furthermore, OFQ significantly increased the intracellular accumulation of ADM in K562/ADM cells and percentage apoptosis in K562/ADM cells. OFQ down-regulated the level of P-gp time-dependently, while the level of Fas and FasL were up-regulated. There were evidently significant differences compared with the control (P < 0.01). After treating K562/ADM cells with OFQ (1 x 10(-7) mol x L(-1)) and ADM (20 microg x ml(-1)) for 48 hours, the cells showed apoptotic nuclear fragmentation, which was characterized by the appearance of a DNA ladder pattern in genomic DNA gel electrophoresis. CONCLUSION: OFQ can reverse the ADM resistance of K562/ADM cells. The mechanism involves OFQ up-regulating the expression of Fas/FasL, down-regulating the level of P-gp, and decreasing the intracellular level of calcium in K562/ADM cells.

Ethanol reduces p38 kinase activation and cyclin D1 protein expression after partial hepatectomy in rats.

BACKGROUND/AIMS: Chronic ethanol consumption inhibits liver regeneration. We examined the effects of chronic ethanol consumption on two mitogen-activated protein kinases in relation to induction of cell cycle proteins after partial hepatectomy (PH). METHODS: Male Wistar rats were ethanol-fed (EF) or pair-fed (PF) for 16 weeks before PH. Hepatic activation of extracellular signal regulated kinase (ERK)1/2, p38 kinase and expression of cyclinD1, cyclin-dependent kinase-4 (cdk4) and proliferating cell nuclear antigen (PCNA) were studied. RESULTS: In PF rats, PH-induced p38 activation was evident at 2h and was maximal at 12h. There was a close temporal relationship between p38 activation, cyclin D1 and PCNA expression. Alcohol exposure reduced p38 activation, cyclin D1 and PCNA, each by approximately 50%. ERK1/2 activation occurred during the first 2h post-PH in both EF and PF rats, and there was no later increase in PF rats. In vivo inhibition of p38 suppressed PCNA expression whereas the effect of ERK1/2 inhibition was inconsistent. CONCLUSIONS: p38 kinase activation is linked temporally with cyclin D1 expression after PH and appears to exert cell cycle control in the adult liver. p38 signaling also appears to be a target for the inhibitory effect of chronic alcohol on liver regeneration.

Endothelin-1 is a potent regulator in vivo in vascular calcification and in vitro in calcification of vascular smooth muscle cells.

We observed changes of endothelin content and endothelin mRNA in vivo in vascular calcification and in vitro in calcification of vascular smooth muscle cells to explore the role of endothelin in vascular calcification. Calcification model in vivo was induced by administration of Vitamin D(3) plus nicotine. Calcification of vascular smooth muscle cells (VSMCs) was induced by beta-glycerophosphate. Endothelin content was measured by using radioimmunoassay. Endothelin mRNA amount was determined by using competitive quantitative RT-PCR. The results showed that calcium content, 45Ca(2+) uptake and alkaline phosphatase (ALP) activity were increased in calcified VSMCs, compared with controls, but were decreased, compared with calcified VSMCs plus BQ123 group. The endothelin content in the medium and endothelin mRNA in VSMCs were elevated by 35 and 120% (P<0.05), respectively, compared with those normal VSMCs. Calcium content, 45Ca(2+) accumulation and ALP activity in calcified arteries increased by 5.0-, 1.4-, and 1.4-fold. The endothelin levels in plasma and aorta as well as the amount of endothelin mRNA in calcified aorta were increased by 102, 103, and 22%, respectively, compared with control group. However, calcium content, 45Ca(2+) uptake and ALP activity in VDN plus bosentan group was 33, 36.7, and 40.4% lower than those in VDN group. These results indicated an upregulated endothelin gene expression as well as an increased production of endothelin in calcified aorta and VSMCs with BQ123 and bosentan significantly reducing vascular calcification. This suggested that endothelin might be involved in pathogenesis of vascular calcification.

Inhibition of taurine transport by high concentration of glucose in cultured rat cardiomyocytes.

Cultured rat cardiomyocytes were treated with 10, 20, and 30 mmol/L glucose and 30 mmol/L glucose plus protein kinase C (PKC) inhibitor, Chelerythrine. In the 20 and 30 mmol/L glucose-treated cells, taurine contents reduced by 15% and 27% (P<.05), respectively, taurine transporter (TAUT) mRNA levels reduced by 47% and 64% (P<.05), respectively, and cysteine sulfinate decarboxylase (CSD) mRNA reduced slightly, but not significantly. Time-dependent taurine uptakes reduced in the 10, 20, and 30 mmol/L glucose-treated cells, and time-dependent taurine release reduced in the 30 mmol/L glucose-treated cells. The Vmax of taurine transport decreased by 18%, 30%, and 35% (P<.05) in the 10, 20, and 30 mmol/L glucose-treated cells, respectively, while Km of taurine transport remained unchanged. When PKC inhibitor, Chelerythrine, combined with 30 mmol/L glucose was treated with the cells, the lowered taurine content, taurine uptake, taurine release, and Vmax of taurine transport caused by 30 mmol/L glucose were eliminated. These results demonstrate that high glucose considerably and specifically decreases intracellular taurine content, taurine transport activity, and TAUT mRNA, possibly through PKC-mediated transcriptional and posttranslational pathways.

Changes in amount of ADM mRNA and RAMP2 mRNA in calcified vascular smooth muscle cells.

This work was aimed to explore the changes and significance of adrenomedullin (ADM) mRNA and receptor activity modifying protein 2 (RAMP2) mRNA in calcified vascular smooth muscle cells (VSMCs). Calcification of cultured rat VSMCs was produced by incubation with beta-glycerophosphate. Content of ADM released by VSMCs was measured by radioimmunoassay (RIA). The amount of ADM mRNA and RAMP2 mRNA was determined by competitive quantitative RT-PCR. The intracellular calcium content, alkaline phosphatases activity and cellular (45)Ca(2+)-uptake were determined. The results showed that the content of calcium, (45)Ca(2+)-uptake and alkaline phosphatases activity in calcified VSMCs were increased by 118%, 174% and seven-fold (all P<0.01), respectively, compared with control VSMCs. Content of ADM in medium was increased by 99% (P<0.01). Furthermore, it was found that the amount of ADM mRNA and RAMP2 mRNA in calcified cells was elevated by 78 and 56% (all P<0.05), respectively, compared with control. The elevated levels of RAMP2 mRNA were in positive correlation with ADM mRNA (r=0.76, P<0.05) in calcified VSMCs. In conclusion, calcified VSMCs generated an increased amount of ADM, and up-regulated gene expressions of ADM and RAMP2.